This invention relates to the production and purification of a recombinant protein for use as a diagnostic tool and as a vaccine against Dengue virus.
Dengue (DEN) viruses are human pathogens with a significant threat to world health. These viruses are estimated to cause several hundred thousand cases of dengue fever, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) annually (Shope, R. E. In: The Togaviruses. Schlesinger, R. W. (Ed.) Academic Press, New York. 1980, pp. 47-82; Monath, T. P. In: The Togaviridae and Flaviviridae, Schlesinger, S. and Schlesinger, M. J. (Eds.) New York and London, 1986, pp. 375-440; Halstead, S. B. Bull. W.H.O. 1980, 58, 1-21; Halstead, S. B. Am. J. Epidemiol. 1984, 114, 632-648) The complete content of all documents cited herein are hereby incorporated by reference. Dengue viruses are members of the family Flaviridae and are transmitted by Aedes mosquitoes (Halstead, S. B. Science 1988, 239, 476-481). There are four serological types, DEN-1, DEN-2, DEN-3 and DEN4, distinguishable by complement-fixation assays (Sabin, A. B. and Young, I. A. Proc. Soci. Exp. Biol. Med. 1949, 69, 291-296), virus plaque-reduction neutralization tests (Russell, P. K. and Nisalak, A. J. Immunol. 1967, 99, 291-296) and immunoassays using monoclonal antibodies (MAbs) (Gentry, M. K. et al. Am. J. Trop. Med. Hyg. 1982, 31, 548-555; Henchal, E. A. et al. Am. J. Trop. Med. Hyg. 1982, 31, 830-836).
Dengue viruses are composed of a single-stranded RNA molecule of positive polarity (messenger sense) which is contained within a nucleocapsid composed of capsid (C) protein. The capsid is surrounded by a lipid envelope about 50 nm in diameter in which are embedded the envelope (E) glycoprotein and the matrix (M) protein. Both the structural and nonstructural (NS) proteins are encoded by a single, long open reading frame of about 10.5 kilobases arranged as follows: C-PreM/M-E-NS1-NS2A-NS2B-NS3-NS4A-NS5 (Rice, C. M. et al. Science 1985, 229, 726-733; Wengler, G. et al. Virology 1985, 147, 264-274; Castle, E. et al. Virology 1986, 149, 10-26; Zhao, B. et al. Virology 1986, 155, 77-88; Mason, P. W. et al. Virology 1987, 161, 262-267; Mackow, E. et al. Virology 1987, 159, 217-228; Sumiyoshi, H. et al. Virology 1987, 161, 497-510; Irie, K. et al. Gene 1989, 74, 197-211).
Attempts to prevent DEN virus infection have focused on the production of a vaccine which would protect against all four serotypes. However, despite more than 50 years of effort, safe and effective dengue virus vaccines have not been developed. Candidate vaccines currently being tested fall into two categories: live attenuated dengue virus vaccines and subunit vaccines, each with its own drawbacks.
Live attenuated virus vaccines have been demonstrated to be either under-attenuated (cause disease) or over-attenuated (fail to immunize). Even an optimally-attenuated live virus vaccine can revert to a virulent (disease-causing) form through mutation. Live dengue viruses are also sensitive to heat, making it difficult and costly to maintain the vaccine in some tropical and subtropical countries where the vaccine may be needed most.
Recombinant subunit vaccines have the advantage of eliminating the risk of infectivity and greater chemical stability. However, the subunit vaccines of flavivirus structural and NS proteins produced in expression vectors including baculovirus, vaccinia virus and E. coli reported so far elicit only low titers of neutralizing antibody and are difficult to produce in large quantities and pure form (Putnak, J. R. et al. Virology 1988, 163, 93-103; Putnak, J. R. et al. Am. J. Trop. Med. Hyg. 1991, 45, 159-167; Zhang, Y. M. et al. J. Virol. 1988, 62, 3027-3031; Lai, C. J. et al. In: Vaccines, Modern Approaches to New Vaccines Including Prevention of AID S (Eds. Lerner, R. A. et al.), Cold Spring Harbor Laboratory Press, New York, 89, 1989, pp. 351-356; Bray, M. et al. J. Virol. 1989, 63, 2853-2856; Bray, M. and Lai, C. J. Virology 1991, 185, 505-508; Men, R. et al. J. Virol. 1991, 65, 1400-1407; Mason, P. W. et al. Virology 1987, 158, 361-372; Mason, P. W. et al. J. Gen. Virol. 1989, 70, 2037-2049; Mason, P. W. et al. J. Gen. Virol. 1990, 71, 2107-2114; Murray, J. M. et al. J. Gen. Virol., 1993, 74, 175-182; Preugschat, F. et al. J. Virol. 1990, 64, 4364-4374).
Both the envelope (E) and the nonstructural protein 1 (NS1) are candidates for recombinant, subunit vaccines against DEN virus. The E glycoprotein is the major surface protein of the virion. It functions in virion attachment to host cells and it can be detected by its ability to hemagglutinate goose erythrocytes. As an antigen, it contains virus-neutralizing epitopes (Stevens, T. M. et al. Virology 1965, 27, 103-112; Smith, T. J. et al. J. Virol. 1970, 5,524-532; Rice, C. M. and Strauss, J. H. J. Mol. Biol. 1982, 154, 325-348; Brinton, M. A. In: Togaviridae and Flaviridae. Schlesinger, S. and M. J. Schlesinger (Eds.), M. J. Plenum, New York, 1986, pp. 327-365; Heinz, F. X. Adv. Virus Res. 1986, 31, 103-168; Westaway, E. G. Adv. Virus Res. 1987, 33, 45-90; Hahn, Y. S. et al. Arch. Virol. 1990, 115, 251-265). Neutralizing antibodies, believed to correlate with protection, and hemagglutination-inhibiting (HI) antibodies develop following natural infection. Mice immunized with purified DEN-2 E antigen develop neutralizing antibodies and are protected against lethal virus challenge (Feighny, R. J. et al. Am. J. Trop. Med. Hyg. 1992, 47, 405-412).
Recombinant DEN proteins have been produced using the baculovirus system for the purpose of developing a vaccine. Results have been variable and sometimes disappointing. Several stategies have been used to produce the DEN E protein in the baculovirus system. One strategy used a truncated gene to produce the E protein without the hydrophobic transmembrane segment of the carboxy terminus. The purpose of this approach was to promote secretion and solubility of the protein. Proteins produced in this manner were minimally immunogenic in mice (Putnak, R. et al. Am. J. Trop. Med. Hyg., 1993, 45: 159-167; Zhang, Y. M. et al., J. Virol., 1988, 62: 3027-3031). Another strategy used a polygene that encoded the capsid, premembrane and two nonstructural proteins, C-prM-E-NS1-NS2 (Delenda et al. J. Gen. Virol., 1994, 75: 1569-1578). This construct produced the full length E protein by cleavage of the polyprotein. Neutralizing antibody to the full length E protein was not elicited by that product although protection was induced. The complex nature of the construct precludes an analysis of the reason for protection in the absence of neutralizing antibody but the presence of NS1 in the construct was speculated to have induced the protective response. Another strategy employed a construct that contained a polygene encoding C, preM and a truncated E protein (Deubel et al. Virology, 1991, 180: 442-447). Although the truncated E reacted with some E-specific monoclonal antibodies (mAbs), reactivity was weaker than that obtained with native virus.
Therefore, in view of the problems with the presently available vaccines discussed above, there is a need for a DEN vaccine that elicits very high titers of neutralizing antibody, provides protection against the disease, has no possibility of infectivity to the immunized host, can be produced easily in pure form, and is chemically stable.
The present invention is directed to a subunit vaccine that satisfies this need. The recombinant DEN virus subunit vaccine of the present invention comprises the full dengue virus envelope protein, expressed in baculovirus and capable of self-assembing into a particle. Dengue envelope protein has been expressed in the baculovirus system by others. The previously produced products were poorly immunogenic when tested in animals. None of the previously made products are known to form particles. The protein is expressed and purified as a particle composed of multiple dengue envelope protein molecules. Particles are more immunogenic than soluble proteins, possibly because they can crosslink cell surface immunoglobulins on B cells. The envelope protein particle of the present invention is produced in baculovirus in large quantities and in pure form, elicits high titers of neutralizing antibody and is protective against the disease in the immunized animal.
The present invention describes the production of the DEN envelope protein particle by cloning the complementary DNA (cDNA) sequences encoding the envelope protein fragment into an expression vector such that the recombinant dengue protein can be expressed. The recombinant protein is produced in baculovirus, isolated and purified as a particle which is antigenic, reactive with dengue virus-specific and monoclonal antibodies and capable of eliciting the production of neutralizing antibodies when inoculated into mice. The administration of this recombinant subunit vaccine is demonstrated to protect mice, an accepted animal model, against morbidity and mortality following challenge with live dengue virus.
Therefore, it is an object of the present invention to provide a DEN 2 cDNA fragment encoding the full envelope glycoprotein, said gene containing 1485 nucleotides plus 93 adjacent upstream sequences and extending from 844 to 2422 of the viral genome and is useful as a diagnostic agent and a naked DNA vaccine.
It is another object of the invention to provide a recombinant vector designed to produce the recombinant DEN envelope protein for use as a vaccine and as a diagnostic agent.
It is still another object of the invention to provide a purified DEN envelope protein particle useful as a vaccine against DEN disease and for detecting the presence of said disease in a suspected patient.
It is another object of the present invention to provide a method for the purification of recombinant DEN envelope protein particle for use as a vaccine or as a diagnostic tool.
It is yet another object of the invention to provide a DEN virus vaccine effective for the production of antigenic and immunogenic response resulting in the protection of an animal against dengue virus disease.